Recent x-ray crystallographic work discloses that the catalytic functions and substantial protons of the active center topologies of vertebrate chymotrypsin and bacterial subtilisins are indistinguishable despite their distinctly different biological sources. Methyl N-acetyl-(S)-phenylalaninate is a good substrate for chymotrypsins and subtilisins. Although the aromatic ring of this substrate doubtless is important in substrate binding, in subtilisin BPN'- and alpha-chymotrypsin-catalyzed hydrolyses of methyl N-acetyl-(S)-phenylalaninate the alpha-acetamido function is also required to achieve efficient, stereochemically selective hydrolysis. It is not known how the alpha-acylamido effect comparatively influences the hydrolysis of activated ester substrates of chymotrypsins and subtilisins. Chemical evidence in favor of or against this interaction might prove useful in defining the limits within which analogies among the subtilisins and chymotrypsins are justified. To initiate the investigation we propose to compare the structural and stereochemical specificities of alpha- and gamma-chymotrypsins and subtilisins BPN' and Carlsberg towards p-nitrophenyl N-acetylphenylalaninate and derivatives in which the alpha-acetamido group has been removed or replaced by alpha-acetoxy. As a means of further probing this aspect of the proteinase specificities, the sensitivity of the chymotrypsins and subtilisins to the nature of the same alpha-substituents in a series of p-nitrophenyl esters of the semirigid indan-2-carboxylic and 1,2,3,4-tetrahydro-2-naphthoic acids will be determined. The latter experiments are considered of particular interest since the geometry of one possible low energy conformer of the locked substrate p-nitrophenyl (S)-2-acetamido-1,2,3,4-tetrahydro-2-naphthoate corresponds quite precisely to that postulated as the reactive conformation of N-acyl-(S)-phenylalanine-derived covalent inhibitors and virtual substrates at the active sites of alpha-chymotrypsin and subtilisin BPN'. Consequently, identification of an alpha-acylamido effect on hydrolysis of the flexible substrates and their rigid models could support the concept of convergent evolution for the chymotrypsins and subtilisins.